In recent years, different types of nucleic acid have been studied for treatment of human diseases. The therapeutic nucleic acids are expected to be developed into a new generation of gene therapy, including DNAs, RNAs, their chemically modified structures and mimics. Among them, RNAs include small interfering RNAs (siRNAs), microRNAs (miRNAs), RNA aptamers, small ligand RNAs (sliRNAs), etc; DNAs include plasmids, etc. These nucleic acids act through a variety of mechanisms. For example, siRNAs and miRNAs regulate the expression of intracellular specific genes by means of a process termed RNA interference (RNAi). When siRNAs or miRNAs are introduced into cytoplasm, double-stranded siRNAs or miRNAs bind to specific intracellular proteins to form RNA Inducing Silencing Complex (RISC). After unwinding of siRNAs in RISC, siRNAs recognize and bind to mRNAs by the mechanism of sequence complementary, leading to cleavage of the mRNAs and down-regulation of the gene expression. Therefore, RNAi provides a potential gene therapy, via complementation with target gene and inhibition of the expression of mRNAs encoding proteins.
The applications of RNAi are extremely broad, since siRNAs or miRNAs targeting a certain protein can be de novo chemically synthesized. A large number of researchers have reported that siRNAs can specifically down-regulate target protein in both in vitro and in vivo model systems. To date, dozens of siRNAs drug candidates are under clinical evaluation.
However, two problems currently faced by siRNAs and other potential therapeutic nucleic acids are, the first, low resistance to the degradation of ribonucleases in cytoplasm, the second, limited ability to cross cell membrane, gain access to intracellular compartment, and bind RISC complex, when naked-siRNAs or miRNAs are administered systematically. These nucleic acids can be stabilized by introduction of chemically modified nucleotides, for example, phosphothioate group-modified nucleotides. Nevertheless, these chemical modifications can only provide limited protection from nuclease degradation, and may compromise the activity of the nucleic acids. Intracellular delivery of therapeutic nucleic acids can be facilitated by various carrier systems, such as polymers, cationic liposomes or chemical modification, for example covalent linkage with cholesterol molecules. Even though, there is still a need to further improve the delivery systems, so as to enhance their in vivo stability and transmembrane activity, as well as to reduce the adverse effect of chemical modifications.
In serum or within the cells, the primary issue faced by therapeutic nucleic acids is stability. In the presence of endo-ribonucleases or exonuclease, nucleic acids are readily to be degraded, with a very short half-life (Zelphati O, et al, Antisense. Res. Dev. 1993(3):323-338). This problem has been partially overcome by the introduction of chemical modified nucleotides, for example modifications at the phosphodiester linkage, at the nucleotide base or at the sugar. Although these modifications decrease siRNAs degradation in serum or within the cells, limitation still exists and the stability problem has not been completely solved.
On the other hand, limited activity to penetrate cell membranes is another problem for the development of therapeutic nucleic acids. To improve this situation, liposome-based carrier systems have been employed to facilitate the delivery of chemically modified or unmodified therapeutic nucleic acids. Numerous studies have reported that cationic liposomes can be used to encapsulate siRNAs, thereby forming liposome-nucleic acid formulation. The delivery activities of these liposome-nucleic acid formulations have been extensively verified in vivo and in vitro.
Despite recent progress, there remains a need in the art to improve the compositions of the liposome-nucleic acid formulation, so as to enable them suitable for general therapeutic applications. Preferably, these compositions would encapsulate nucleic acid with high-efficiency, have high drug: lipid ratios, protect the encapsulated nucleic acid from degradation by intracellular nuclease, suitable for systemic delivery, and facilitate intracellular delivery of the encapsulated nucleic acid. In addition, these liposome-nucleic acid formulations should be well-tolerated and provide appropriate therapeutic index, such as an effective dose of the nucleic acid and no significant toxicity to the patient. The present invention provides such liposome formulations, methods of preparing the liposome formulations, and methods of using the liposome formulations to introduce nucleic acids into mammal cells, for the treatment of human diseases.